Isolation and analysis of fluP, a gene associated with hyphal growth and sporulation in Aspergillus parasiticus

Abstract.

Aflatoxins (AF) are polyketide-derived mycotoxins that frequently contaminate food and feed crops, causing health risks to animals and humans. The fluP gene was cloned by screening an Aspergillus parasiticus genomic DNA library with a cDNA probe encoding part of a polyketide synthase (PKS), the 6-methylsalicylic acid synthase (MSAS) from Penicillium patulum. FluP was hypothesized to function as a PKS in AF biosynthesis. The predicted amino acid sequence of FluP demonstrated a high degree of identity to MSAS (55%), moderate identity to another fungal PKS protein encoded by wA from A. nidulans (22%) and low identity (<5%) to fungal fatty acid synthase (FAS) proteins. Disruption of fluP in A. parasiticus resulted in the loss of fluP transcript, a 3- to 4-fold reduction in hyphal growth rate, the appearance of a fluffy, cotton-like hyphal morphology, reduction or elimination of asexual spores and spore-bearing structures, and a twofold reduction in aflatoxin accumulation. Removal of selective pressure on fluP knockout transformants resulted in frequent reversion (10%) to the wild-type genotype and phenotype, establishing a direct link between gene disruption and the associated phenotype. The data suggest that fluP encodes a novel PKS associated with hyphal growth and cell development (sporulation), whose activity indirectly influences aflatoxin accumulation in A. parasiticus.